The utility of the baculovirus cell expression system for investigating Ca 2+-ATPase and phospholamban regulatory interactions was examined. cDNA encoding the canine cardiac sarco(endo)plasmic Ca 2+-ATPase pump (SERCA2a) was cloned for the first time and expressed in the presence and absence of phospholamban in Spodoptera frugiperda (Sf21) insect cells. The recombinant Ca 21 pump was produced in high yield, contributing 20% of the total membrane protein in Sf21 microsomes. At least 70% of the expressed pumps were active. Coexpression of wild-type, pentameric phospholamban with the Ca 2+- ATPase decreased the apparent affinity of the ATPase for Ca 2+, but had no effect on the maximum velocity of the enzyme, similar to phospholamban's action in cardiac sarcoplasmic reticulum vesicles. To investigate the importance of the oligomeric structure of phospholamban in ATPase regulation, SERCA2a was coexpressed with a monomeric mutant of phospholamban, in which leucine residue 37 was changed to alanine. Surprisingly, monomeric phospholamban suppressed SERCA2a Ca 2+ affinity more strongly than did wild- type phospholamban, demonstrating that the pentamer is not essential for Ca 2+ pump inhibition and that the monomer is the more active species. To test if phospholamban functions as a Ca 2+ channel, Sf21 microsomes expressing either SERCA2a or SERCA2a plus phospholamban were actively loaded with Ca 2+ and then assayed for unidirectional 45Ca 2+ efflux. No evidence for a Ca 2+ channel activity of phospholamban was obtained. We conclude that the phospholamban monomer is an important regulatory component inhibiting SERCA2a in cardiac sarcoplasmic reticulum membranes, and that the channel activity of phospholamban previously observed in planar bilayers is not involved in the mechanism of ATPase regulation.